Diagnostic agent for detection of primary hepatoma

ABSTRACT

A DIGANOSTIC AGENT FOR PRIMARY HEPATOMA COMPRISING ANTI-A1-FETOGLOBULIN, AND A PROCESS FOR THE PREPARATION OF THE DIAGNOSTIC AGENT FOR PRIMARY HEPATOMA WHICH COMPRISES THE STEPS OF DISSOLVING THE ANTI-A1-FETOGLOBULIN TOGETHER WITH A SUPPORT MEDIUM AND ALLOWING THE SAME TO SOLIDIFY IN A DIFFUSED CONDITIONS.

United States Patent nr oNos rrc AGENTTORDETECflTONT PRIMARY HEPATOMA I v Hidetaka Nagai, Tokyo, Eiichi Sato, Funabashi, and FlljlO KobayashLjIfolryo, Japan, assignors to KowaColnpauy, Ltd., Naka ku', Nagoya, Japan v- No Drawing' Filed Dec. 17, 1970, Ser. No. 99,312- Claims priority, application Japan, Dec. 18,1969, 44/101,870; Feb. 19, 1970, 45/ 14,335; July 29, 1970, "45/ 66,238; Aug. 28,1970, 45/75,452 Int. Cl. G01n 31 /02, 33/16 U.S. 'Cl.424--.8 I .1 6 Claims ABSTRACT OF THE- DISCLOSUR A diagnostic [agent for primary hepatoma' comprising anti-al fetoglobulin, and a process for the preparation of he agno t gen f r p ma y pato 'fw ch mprises the steps of dissolving the anti-al-fetoglobulin together with a support medium and allowing the same to solidify in a diffused condition.

BACKGROUND OF THE INVENTION The instant-invention relates to diagnostic agents which are highly useful and highly sensitive fo'r the clinical diagnosis of primaryv hepatoma as distinguished from 3,819,822 Patented June 25, 1974 ICC It is another object of the invention to provide a purified grade of anti-al-fetoglobulin which is important in the preparation of the diagnostic agents of this invention.

It is still another object to provide a purified grade of al-fetog'lobnlin which is important in the preparation of to be. adsorbed onto a carrier medium and which is then dispersed in a dispersion medium.

Another important feature of this invention lies in the method used in the preparation of such a purified grade of anti al-fetoglobulin. Thus, the ascites or serum derived I fromapat'ienthaving a hepatoma or the serum of the metastatic and, other liver disease. More particularly, the

invention relates to new serological diagnostic agents for the determination of primary-hepatoma, which are based on anti wl fetoglobulin. This invention also relates to methddsf'for the'preparation of said 'serological diagnostic agents."- Furthermore, this-invention relates to diagnostic methods which depend upon the use of said diagnostic a ent.

a 0riginally,.it-was found that'the serum ofthe cattle fetus "contained-"a type of cal-globulin which was designated al-fetoglobulin (Fetuin) [K. O. Pedersen, Nature 154, 575 (1944)]. It was then discovered that thisparticular substance also occurredinthe se'ra of fetuses of different animal species and wasspecific to various "species. Those findings were followed by the discovery that the same substance also occurred inthe serum of the'human fetus, C. G. Bergstrand, et al., J. Clin..& Lab. Invest. 8

174 (195.6). Recently, it was further demonstrated by immunological precipitin reactions, that ul-fetoglobulin occurs inthe 'sera of patients having primary hepatoma [Yu. S. Tatarinov, Vop; Med. Khim. 11 2)--20 ('1'9 65) and others]. It has thus been suggested thatthe presence or absence of al-fetoglobulin may provide a test for primary hepatoma. Over the succeeding years, we conducted extensive'research into the basic mechanism of these cancers on the strength of finding that the metabolism of-the human being afflicted with the cancerous diseases are-analogous tothat of the fetus, and that al-fetoglobulin is closely related to primary' hepatoma. Qurj effort has been largely concentrated on the development of improved diagnostic agents for clinical use.

. SUMMARY OF THE INVENTION" The effort" has now been repaidby the discovery of a quick and accuraternethod for the diagnosis and quantitative determinationof' those cancers, whichldofnot involve the use of-complicated chemical-operations and costly, intricate equipmentf n is-an object or this invention as provide a diagno tic agent which offers afhiigh'idetection sensitivity forthosediseases, a reduced incidence of doubtful posit iv and a high degreeof-quantitative its,

human fetus is used to immunize an animal. A blood sample is taken from the animal. From this blood sample the serum is separated and the antiserum is absorbed with normal human serum, or with an insoluble immunoadsorbent, obtained by treating normal human serum with, for example, glutaraldehyde. Alternatively, the above ascites or serum is preliminarily purified by usual protein fractionation and the resulting al-fetoglobulin fraction is used to immunize an animal. A blood sample is then taken from the animal and the serum is separated.

Another important feature of the invention lies in the method of preparing the purified grade of al-fetoglobulin. The ascites or serum of a patient having a primary hepatoma is dialyzed against acrinol and precipitated. The dialyzate is purified by a protein fractionation technique. Since the first suggestion was made,- as "mentioned above, that the presence or absence of al-fetoglc ibulin' might be used in the discriminatory diagnosis of hepatoma, a number of workers have attempted: immunodiagnosis of primary hepatoma. To this day, however, no

satisfactory diagnostic agent has been discovered, and,

-- at present, the immunodiffusion and irnrnuno electrgphoretic techniques, both being versatile methods u s eful in many immunodiagnostic applications, are. :beingutili'zed by analogy. Immunodiffusionmay be classified into single and double diffusion techniques. The single diffusion technique is a diagnostic method in which a tube containing the antigen, the antibody and'rdiifusion' medium in three distinct layers gives a precipitin band in" the middle layer comprising'said diffusion medium at the level corresponding to the optimum antigen-antibody ratio. This level is used as a diagnostic measure and'is generally known as Oudins method. The double diffusion technique, known as Quchterlonys method, is such that the antibody is placed in a central well and the antigen is placed in a plurality of satellite wells, each of said wells having been bored in an agar layer on a plate. A precipitin ring appears in the position corresponding to the optiiiium antigen-antibody ratio, as has just been described above for the single diffusion technique, and their precipitin ring is used for diagnosis. Oudins method, in which a precipitin ring is developed at a level which is determined -by the ditfu'sion'coefiicients and the concentrations of the antigen'and antibody, is advantageous for the quantita- 'tion of the antigen. On the other hand, Ouchter-lonys method proves more useful when it is used to discriminate between different antigens.

Immune-electrophoresis is a combination of ordinary zone electrophoresis and the above-mentioned immunoditfusion. Thus, if the antibody is positioned in parallel with the direction of migration of the antigen, which has been linearly fractionated, a precipitin ring is developed in a location corresponding to the optimum ratio, as has been described above, and this precipitin ring is used as a diagnostic measure. This method is excellent in reproducibility. I

However, those prior art diagnostic methods are different from the methods developed specifically for the discriminatory diagnosis of the subject disease of this invention, and although they may be used, each of those methods have many disadvantages. For instance, those methods are characterized by a high incidence of doubtful positive results. They require expensive equipment and involve complicated and time-consuming procedures which are incidental or essential to the necessary diagnosis. Thus, a more specific and simple method has been desired in order that a large number of patients'may be diagnosed in a limited time.

The studies made by us in an effort to overcome those numerous disadvantages have led to the finding that by dissolving anti-al-fetoglobulin together with a support medium and allowing it to solidify in a dispersed state or" causing anti-al-fetoglobulin to be adsorbed on a carrier medium and, as such, dispersed in a dispersion medium, one may obtain a diagnostic agent using the ascites or serum of a patient directly as the specimen to be arranged, without resorting to any complicated procedures. We have also succeeded in the preparation of diagnostic agents which remain stable even after prolonged storage and are least liable to give doubtful positive results and capable of providing an advantageous test for pri mary hepatoma. This invention is the culmination of the foregoing discovery and development. Many other ad-' vantages and features of the instant invention will become apparent as the following description of the invention proceeds.

DETAILED DESCRIPTION OF THE INVENTION The different embodiments of this invention will now be discussed in greater detail.

(1 The following description is directed to the preparation of adiagnostic agent of this invention, wherein the antibody, anti-al-fetoglobulin, is dissolved together with a support medium and the solution is allowed to solidify in a diffused condition.

Thus, this particular diagnostic agent is prepared by using the antigenic al-fetoglobulin to immunize an animal. The resulting anti-serum is dissolved together with a support medium and, if desired, a surfactant, coloring agent, and/or preservative. This solution is allowed to solidify in the diffused condition. I

' The support medium used in the practice of this invention is one which has a high diffusion coefiicient in terms of viscosity. For purposes of discrimination convenience, the medium should be characterized by a satisfactory degree of clarity. Among the media that satisfy the above conditions are agar, dextrin, starch, pectin and other polysa'ccharides, and various proteins including gelatin and the like. Preferably, agar, synthetic polyacrylamide gel andstarch are employed. The surfactant is used in the practice of this'invention for the purpose of reducing the percentage of doubtfulpositives attributable to hypercholesteric sera in clinical applications, and may be selectedfrom the group consisting of anionic surfactants suchas sodium oleate, sodium laurylsulfate, sodium octyl-.- sulfate, sodium stearyl sulfate, sodium alkylbenzene sul-.,.

fonate, sodium dialkylsulfosuccinate, etc., cati oniesurfactants such as dodecylamine acetate, benzyldime'thyl alkylammonium halide, cetylpyridinium ha1ide,'e tc., and nonionic surfactants such as poly'oxyethylene fatty acid" esters, polyoxyethylenesorbitan fatty acid esters, polyoxyethylene (higher) alcohol ethers, polyoxyethylenalkylp Q 4 aryl ethers, polyoxyethylene castor oil derivatives, etc., as well as lecithin, saponin, taurocholic acid and the like. Among the preferred surface active agents which provide particularly beneficial results include polyoxyethylenesorbitan fatty acid esters (Tween type) which are nonionic. A colorant may be used to facilitate the diagnosis. The colorant may, for example, be Evans blue, toluidine blue, brilliant green, brilliant blue 'FCF, alcian blue, patent blue, trypan blue amido black 10B or Panceau 3R. Among those colorants, patent blue, and trypan blue are particularly advantageous. A preservative may be added including the various phenolic compounds, such as cresol, chlorothymol, thymol, parahydroxybenzoic acid ester, etc.; organic mercury compounds, such as thimerosal, phenylmercury "acetate, phenylmercury borate, etc.; chlorobutanol and other alcohols, sodium azide, and the like. The mode of practice of the instant invention will hereinafter be described in detail.

A buffer solution of predetermined pH and ionic strength is prepared for the purpose of stabilizing the diagnostic agent. This buffer solution is preferably a tris-HCl-saline or glycine-sodium chloride buffer, although citrate, phosphate, borate and barbiturate buffers may likewise be employed. The pH of this solution may range from weakly acidic to weakly alkaline; that is to say, from pH 6.5 to pH 8.5. The optimum pH lies somewhere between pH 7.2 to pH 8.2. The above buffer solution is maintained in a container at a temperature of preferably from 50 C. to 56 C., and the support medium is dissolved in the solution. It is preferable that the support medium be added to a final concentration of l to 15 w./v.%.

The anti-serum prepared by the procedures hereinbefore described is dissolved in the same buffer as above to a final concentration of l to 10 w./v.%, and the result ing solution is admixed with the support-buffer solution prepared above. The surface active agent and coloring agent are added to final concentration of 0.01 to-0.20

w./v.% and 0.001 to 0.005 w./v.%, respectively. When" the surfactant used is taurocholic acid, the final concentration should be between 0.2 to 0.5 w./v.%. To the thusprepared composition, the preservative agent is added until a final concentration of 0.1 w./v.% is reached. After sufficiently mixing the composition, an aliquot is poured into a suitablevessel, in which it is allowed to solidify. The desired immunodilfusion plate is then obtained by providing wells in the solidified composition, from which the test serum may diffuse. It is to be understood that, in the practice of this invention, the foregoing procedures may be modified within the limits that are obvious to those skilled in the art. l

The vessel-in which the diagnostic agent of this invention is to be filled and stored must be such that it is transparent throughout and can be hermetically sealed to prevent evaporation of moisture. In addition, the vessel should be of a suitable design: to lend itself to easy.

manipulation. An example of a vessel which satisfies these requirements is a colorless, transparent plastic vessel suitable for containing a levelof diagnostic agent up to 2- millimeters. After the foregoing-procedures have been. carried through, the diagnosis and quantitation, accordingto, this invention, can'be conducted with extreme "thre'e" preparations'of the crude, IO-and' I'OO-fold serums.

It""is'oiie"'of"'the. advantages of this technique that since the diagnostid'ag'ent obtained in the above-described manner enables the antigen to be quantitated, the progress or stage of the primary hepatoma can be easily' determined.

(2) The following" description relates to the embodiment of this invention wherein the anti-ul-fetoglobulin, which is the antibody, is'adsorbed on a carrier medium which is then dispersed in a dispersion medium.

To obtain this diagnostic agent, anti-al-fetoglobulin is first adsorbed on a carrier medium, which is then dispersed in a dispersion medium. The carrier medium used in this embodiment of the invention may be selected from among the common adsorbents andion exchange resins, including aluminum-silicateadsorbents, such as kaolin, bentonite, diatomaceous earth, acid clay, etc.; magnesium-aluminum-silicate adsorbents, such as beagum, etc.;"silicic anhydride, acrylic resin adsorbents; polystyrene latex; polyvinyltoluene 1atex;"Bioglass (manufactured by Muromachi'Chemical Industries, Ltd.);" ion exchange resins, such as Amberlite IRA411, IRA93, IR45, I-R4B, :IRA68, and IRCSO (Rohm &- Haas Company); Dowex 44 (Dow Chemical Company), andothers. Since the above carrier medium exerts an influence on the adsorptionand stability characteristics of 'the diagnostic' agent, it is preferably spherical in shape and uniform in grain size. The diameters of the grains are desirably within the range of 0.5 to 3 microns.

To cause anti-al-fetoglobulin to be adsorbed on this carrier medium in accordance with this invention, the carrier medium is first dispersed in a suitable buffer solution and the anti-serum is added to the resulting dispersion, followed by thorough shaking.

For the present purpose, a variety of buffer solutions may be employed. For instance, tris-HCl-saline bufier and sodium chloride-aminoacid buffer and barbiturate buffer, as well as citrate, phosphate and borate buffers can be used to advantage. It is preferable that the above carrier be added in a sufficient amountto give a 'final concentration of 0.2 to 5 w./v.%. The amount of the anti-serum depends upon the concentration of anti-ulfetoglobulin. Generally speaking, the anti-serum is previously dissolved in the above buffer to a final concentration of 1 to w./v.% and in the case of the purified antibody, it is dissolved in the above buflfer to a final concentration of 0.1 to 1 w./v.%. Thesolution is added to the above carrier-buffer, followed by suflicient shaking.

In this manner, the 'anti-o l-fetoglobulin contained an the anti-serum is adsorbed around the carrier to give an anti-m1-fetoglobulin-carrier combination.

This anti-al-fetoglobulin-carrier combination is then separated by, for example, centrifugation and, after being washed with, for example, the same buffer solution as above, is dispersed in a dispersion medium. The dispersion medium isdesirably one of the above-mentioned buffer solutions. In this connection, a suitable dispersing agent is used to ensure a more uniform dispersion of the anti-al-fetoglobulin-carrier combination and a st'abilized maintenance of anti-ul-fetoglobulin in adsorbed condition.

The dispersing agent mentioned above is desirably selected from among albumin, peptone, gelatin, agar, saponin, alginic acid, polyvinylpyrrolidone, polyphosphoric acid, serum and the like. Aside from those dispersing agents, various surface active agents may be employed. Suflicient effects may be achieved by using such an agent in a proportion of from 0.1 to 1 w./v

For added ease of diagnosis, a coloring agent may be incorporated into the diagnostic agent. Suitablecoloring agents include, those colors mentioned above and-particularly patent blue and trypan blue provide satisfactory results. t

Because the diagnostic agent of this invention -is, in many cases, subjected to prolonged storage, it -is advantageous to incorporate a small amount ofapreservative such as-those specified'above; Y

It is to be noted that the anti-al-fetoglobulin-carrier combination is dispersed desirably to a final concentration of 0.5 to 5 w./v.% and, at the last stage, the dispersion is adjusted to pH 6.5-8.5 and, for the best result, to pH 8.2-8.5.

The diagnostic agent thus obtained is superior to that prepared by dissolving the same anti-serum together with a support medium and allowing the same to solidify in a diffused state, since it enables diagnosis in a shorter period of time. Regardless of the diagnostic agent, however, in order to increase the sensitivity of the agent to detection of disease and to lower the incidence of false positive results and establish a high reliability as to quantitation, it is important to obtain a purified grade of anti-u1-fetoglobu1in. To this end, the impurities should be removed as much as possible from the ascites or serum of the patient suffering from a primary hepatoma, or from fatal serum.

(3) The following explanation is relevant to the case wherein the ascites or serum from a patient having a primary hepatoma, or fetal serum is used to immunize an animal, which is then bled, the serum is separated from the blood sample and the resulting anti-serum is absorbed into normal human serum.

The ascites or serum obtained from a patient suffering from a primary hepatoma or serum from a fetus is added to, for example, an equal amount of physiological sal-ine,- followed by the addition of an equal amount of Freunds complete adjuvant (manufactured by Difco). After being emulsified, the system is used to immunize an animal. The animal may be, for example, a rabbit, a horse or an ox. Particularly beneficial is a method wherein a rabbit is subcutaneously injected in a foot pad. Injections are repated at timed intervals and after a sufficient gain has been confirmed in antibody titer, the animal is bled and the serum is separated. The resulting anti-serum is absorbed with normal human serum or with the abovementioned insoluble immunoabsorbent to obtain a specific anti-serum.

The anti-serum is desirably high in anti-ul-fetoglobulin concentration in order to increase the sensitivity of de tection of the disease in question and to-lower the incidence of false positive results. Therefore, --better results are obtained if the above. anti-serum is purified by, for example, ammonium sulfate fractionation.

Since the ascites or serum of a patient having a primary hepatoma, or fetal serum contains various serum proteins, such as albumin, .ry-globulin, etc., in addition to al-fetoglobulin, when animals are immunized with the ascites or serum assuch,. antibodies are found which correspond to those various serum proteins in addition to anti-al-fetoglobulin. It is therefore necessary to absorb this anti-serum with a large quantity of normal human serum. However, the anti-ul-fetoglobulin obtained in this stage has already been highly purified and can be directly dissolved along with a support medium or, alternatively, dispersed in a dispersion medium after being adsorbed on a carrier medium to prepare a satisfactory diagnostic agent.

Preferably, however, the antiserum after the absorption step is further purified.

(4) The following description is directed to the mode of practice, in which the ascites or serum of a patient having a primary hepatoma, or fetal serum, is first purifiedby a protein fractionation technique and the resulting 'ul-fetoglobulin fraction is used to'immunize an animal which is then bled, followed by separation ofthe serum from the blood sample thus collected. Thus, the ascites or serum obtained from a patient suffering from a-' primary hepatoma, or fetal serum, is purified by a conventional serum protein fractionation technique. Suitable known fractionation techniques include Cohns fractionation method, Spiros modified method, ammonium sulfate fractionation ('salting-outL-"gel filtration, column chromatography, isoelect'ric""fractionation, electrophoresis, membrane filtration and the like, which techniques may be employed either singly or in combination.

The thus-obtained al-fetoglo'bulin fraction is dissolved in, for example an equal amount of physiological saline solution, followed by the addition of Freunds complete adjuvant (Difco) to emulsify the same. The animal is then immunized with the above emulsion. Suitable animals for this purpose include such species as rabbits, goats, cattle and horses. Particularly preferred are rabbits, whose foot pads are choice sites for subcutaneous inoculation. Injections are repeated at suitable intervals, with the gain in titer of the antibody being traced constantly. When the. antibody titer is at the maximum, the ear vein is punctured to collect blood samples every other day. The antibody titer reaches its peak value in above 1 to 2 weeks after. injec tion. The serum is separated and absorbed with a small amount of normal human serum or its gel is used to obtain the desired anti-a l-fetoglobulin.

In the practice of this invention, as has hereinbefore been described, only a very small amount of normal human serum is required to absorb the antiserum. For instance, when the serum has not been purified, it is necessary to use at least equal amounts of normal human serum and antiserum. In contrast, in the method of this invention, 0.1 to 0.35 of normal human serum is required per part of antiserum. Furthermore, the antibody titer of the antiserum obtained by the method of this invention, as arranged aganist the standard antigen, is 2 to 8 times as high as that of the crude antiserum.

By the afore-described procedures, a high degree of purification can be accomplished. In addition, the method of this invention avoids many of the disadvantages of the prior art. Thus, the use of a large quantity of normal human serum was not only commerciallydisadvantageous, but was also liable to result in a reduction in antibody titer caused by coprepicitation of anti-ul-fetoglobulin. In'addition, the priorart procedures were not sufiicient to overcome the difiiculty of absorbing anti-a2-macroglobuli'n' and analogous substances. In contradistinction, this invention makes it feasible to obtain a high grade of antiserum with the use of extremely small amounts of normal sera. Furthermore, there is substantially no contamination With anti-a2-macroglobulin andanalogou's substances. A 'further striking fact is that the antibody titer 'of the antiserum obtained by the present method is by far higher"thah'that obtainable by the conventional procedure referred to above. The anti-al-fetoglob'ulinserum which has thus been prepared is then eitherdissolved' in a buffer solution" together with a support medihm-or-adsorbed on a carrier medium and dispersed in a dispersion medium, whereby an excellent diagnostic agent is prepared.

' The purposes of the present diagnostic agent can'generally be fully achieved by using the anti-al-fetoglobulin obtained in the foregoing manner; However,'when'it is desired to obtain an unusually high grade of the diagnostic agent, for example, for academic purposes, it becomes necessary to purify' the al-fetoglobulin to an extreme degree of purity. {Borcedures that will be necessary for the preparation of such high grades of al-fetoglobulin'also fallv within the purveiw of the instant invention. 7

We will no w describe the purification technique wherein the ascites, or serum obtained'from a patient having a primary hepatoma, or serum from ahurnan fetus,is dialyzed against acrinol and'the precipitate arising from the dialylate is purified by the smarter a protein fraction. Thus, the asci'te's or serum from a Pat ent havnig' a" primary hepatoma, or human fetal serum, isdiluted with water to 3' 5 times the initial volume and dialyzedagainst an aqueous solution of 0.1-0,01 w./v.% acrinol. dialysis is desirably carried out against acrinol' and the serum or ascites as an internal phase for about 24 hours. I

This acrinol solution is then centrifuged or otherwise treated to obtain a sediment'which is a fairly purified grade of al-fetoglobulin. However, since this sediment also 'con- 8 tains small amounts of impurities, including albumin, it can be fractionated by conventional protein fractional purification techniques to obtain a purified grade of a1- fetoglobulin. I

Among the applicable protein-fractionation-purification techniques are ammonium sulfate fractionation, dialysis, column chromatography, membrane filtration,.isoelectric fractionation and other techniques, which may be practiced either singly or, if necessary, in combination. The best possible result may be obtained by thefollowing procedures. The aforementioned sediment is first dissolved in, for example,-a physiological saline solution or a buffer solu tion and after the insoluble matter is removed, the solution is dialyzed against a buffer solution (ionic strength: 0.03 to 0.04 M and pH 4.5 to 5.5). The buffer solution may, for example, be acetate buffer, tri-HCl-saline buffer, sodium chloride-glycine buffer, citrate, phosphate or borate buffer. The dialyzate is then run into a column or carboxymethyl-cellulose column (ionic strength 0.03-0.04 M; pH 4.5-5.5) and eluted from 0.03-0.4 M to 0.2 M. In this manner, the desired al-fetoglobulin is eluted from the column, starting at about 0.055 'M. The resulting fractionsv are pooled and a pure grade of al-fetoglobulin is obtained. Aside from carboxymethyl-cellulose, the above column chromatography may likewise be carried out on such other materials as DEAE-cellulose, TEAE-cellulose and the like. This column chromatography is desirably conducted in repetition, i.e., two or more times, at leastone of which.

duction of the diagnostic agent. This -removal not only involves complicated procedures but also leads to considerable reductions in product yields. 7

The following methods are known for the collection and purification of alfetoglobulin. r

(1) Bergstrand and Czar used fetal sera as the raw material and fractionated the same electrophoretically. They found al fetoglobulin in a location which coincides with that of albumin and reported that they had noted a slight difference in molecular weight as determined by the ultra-.-

centrifugatio'n technique [Scand. J. Clin. & Lab. Invest.,

- (2) Davis, et al; passed umbilical sera twice through a column of Sephadex 6-200 and eluted the same with 01 M aqueous solution of sodium chloride. It was reported that they had noticed the concentration peak of al-fetoglobulin'in the fraction coincidental with those of transferrin and albumin, [J. C1in. Invest., 45 (1966)].

(3) Hirai,'et a1. caused'fetal vsera to be adsorbed on a column of DEAE-cellulose (pH 5.5, 0.02 M) and conducted a g'radient'elution aganist 0.2 M, whereupon 1x1 fetoglobulin was eluted as the shoulder of an albumin peak. This fraction was then adsorbed on a column of CM-cellulo'se""(pl-I 4.8 and 0102 M) and a gradient elution conducted against 0.5 M As a result, they obtained three peaks, of which al-fetoglobulin was the third. The latter half of this peak was rechromatographed to obtain a single peak for al-fetoglobulin. (Repeated before the Society of Electrophoretic Researchers on Oct. 1, 1969).

Those reports, however, went no further than to confirm the existence of al-feto globulin or the production of al-fetoglobulin in a crude form, and these methods are not conducive to the obtaining of high grades of al-fetO- globulin. I

Nevertheless, insofaras it is to be used as theraw material for a diagnostic agent forprimary hepatoma in academic research applications, al-fetoglobulin must be of particularly high purity, since its purity has an important influe nce upon the accuracy of the diagnostic agent.

. In view of the foregoing, we conducted extensivestudies to obtain a highlypure grade of ul-fetoglobulin and, particularly, to develop a method for positively removing thecontaminant albumin at low cost. Those studies have ultimately led to the finding that a pure grade of alfetoglobulin can-be obtained by first dialyzing sera or other materials containing al-fetaglobulin against acrinol and then fractionating the dialyzate by procedures which are conventionally followed for fractional purification of proteins. The instant invention is the culmination of the above finding and affords an extremely useful method for the preparation of a high grade of al-fetaglobulin without resorting to complicated procedures.

Clinical data pertinent to the use of the diagnostic agents .of this invention are given below.

Of the diagnostic agents for primary hepatoma according to this invention, the clinical results for diagnosing primary hepatoma with the use of the diagnostic agents of the present invention using a support medium are set forth in Table 1. There were a total of 63 cases, most of which were diagnosed at the National Cancer Center Hospital (Japan) including both inand out-patients. Of these, 12 were primary hepatocarcinomatous .cases (of which 11 cases were established by tissue diagnosis), 15 were metastatic hepatocarcinomatous cases, 15 were hepato-cirrhosis cases, 22 were hepatitis cases, and 1 was a hepatangiomatous case. The antigen quantitation results for the positive cases which showed precipitin rings in the present array and the results obtained by the conventional diagnostic technique are set forth in Table 2.

TABLE 1 Number of- Cases Positive Disease diagnosed cases Primary hepatocarcinoma 12 11 Metastatic h'epatocarcinoma. 15 Hepatocirrhosis 1'5 0 'Hnnatitis 22 0 Hepatoangioma'. 1 0

TABLE 2 Results by COIIVBH- Name ofpatient, Antigen tional initials only Age Sex (#/I1'll.)*** method 23 b 1,600 65 Q 9 60 Q 11 58 b 150 8 0 15,000 1, 0 1 "o 65 b 2,826 19 "b 5 62 300 62 is 1,300 3 '1; 130

*Ascite was used; Sera was used in other cases. "Ouchterlonys method. /ml=0.3 6/1111 It will be seen from the above Table that in those cases which were diagnosed positive with the diagnostic agent of this invention, the conventional diagnostic method failed to identify the disease when the antigen titers were less than 10 a/ml.

For the diagnosis of primary heptamoa using a diagnostic agent prepared with a carrier medium, the following sirnple procedure was used. A drop of the diagnostic agent was placed on a slide glass, where a fresh sample of serum was mixed with the diagnostic agent. When this admixture occurs, a specific agglutination reaction occurs if the patient has a primary hepatoma. As will be seen from the following clinical data, comparison of this particular diagnostic agent and the previously-described diagnostic agents prepared with use of a support medium reveals that all the primary hepatoma prove positive, irrespective of whether sera or ascites are employed.

Support methd Dia. of Sample This band Dilution arranged method mm. factor, times Name of patient,

initials only M. 8.-.; Serum--. 6.0 X8. S. Y- do 5.7 X8. 0. Y -do... 4.0 Original serum. Y.S do 4.2 Do. K.N 0 6.2 X8

K. Y o 4.0 Original serum. A. S. do. 7.3 X8. N. S 6.0 X8. E. S Ascrtes... 12.3 X8. S. Y do 7.5 X8. Normal human..." Se 0.0

NOTE

1. The above data were obtained at the National Cancers Center. 2. The designations of results in the table are negative; weakly positive; positive and strongly positive.

Having now generally described the inventlon, a better understanding can be obtained by reference to certain specific Examples which are provided herein for purposes of illustration only and are not intended to be limiting in any manner.

PREPARATION OF THE DIAGNOSTIC AGENTS WITH A SUPPORT MEDIUM Example 1 To tris-HCl-saline buffer (pH 7.2) is added a refined immunological grade of agar to'a final concentration of 1.5%. The mixture is heated, and the agar is completely dissolved. This solution is maintained at a temperature of C. Separately, rabbit antiserum is absorbed with 0.35 parts of normal human serum to make a final concentration of 3 w./v.% which is mixed with the agar support prepared above. Tween 80, patent blue and sodium azide are added in sufiicient amounts to provide a final concentration of 0.01%, 0.001%, and 0.1%, respectively. After To sodium chloride-glycine buffer (pH 8.2) is added an immunological refined grade of agar to a final concentration of 1.0 w./v.%, followed by heating to 50 C. Separately, horse antiserum is added to the same buffer as above in a sufiicient amount to give a final concentration of 4 w./v.%, followed by heating to 50 C. The two solutions are mixed, and sodium taurocholate is added,

in a sufiicient amount to give a final concentration of 0.3%. Trypan blueand chlorobutanol are then added to the above system until the final concentrations are 0.002.% and 0.3%, respectively. After the additives are dispersed, the system is poured into a vessel. After the system has solidified, wells are bored therein to obtain an immunological agar plate.

Sodium chloride is added to trisor amino acid buifer (pH 6.5-8.5) to prepare an isotonic solution, and the following material are added until the designated final concentrations are reached.

Final Final Final 7 Final cone, cone, cne., conc., Ex. No. Support medium percent Surfactant percent Coloring agent percent Preservatlve percent 3 Synthetic 1.5 poly- 1.5 Sodium oleate. 0.1 Brilliant Green--- 0.002 Thymol. 0.1

acrylamlde gel. 4 Pectin 1.3 Al lryltr i nethylammonium 0.05 Patent Blue 0.001 Phenylmercury acetate.... 0. 02

rom e. 5 Dextrin 1.5 Polyoxyethylenesorbitan 0.1 Alcian Blue 0.003 Sodium nitride 0.1

monolaurate. v 0 Starch 1.0 Saponin 0.03 Trypan Blue 0.001 Phenylmercury borate 0.01 7 Agar 1.2 Sodium myristyl su1tato.-..- 0.15 Brilliant Blue..." 0.004 Thimerosal 0. 01

PREPARATION OF THE DIAGNOSTIC AGENT WITH A CARRIER MEDIUM Example 8 1.5 g. of crushed, purified Amberlite IR45 (1-3;/. in size) is dispersed into milliliters of sodium chlorideglycerine buifer solution (pH 8.2), followed by the addition of an optimal amount of anti al-fetoglobulin serum. The system is shaken at 4 C. for 2 hours, whereby the anti-al-fetoglobulin is adsorbed onto the carrier medium. The sediment Amberlite IR45 coated with the anti-a1- fetoglobulin is centrifuged twice, using the same buffer solution as above to which 0.3 w./v.% albumin and 0.1 w./v.% sodium azide have been added. After washing, the carrier-anti-ul-fetoglobulin is dispersed in about 90 ml. of the same buffer solution and is brought to an exact pH of 8.2. An additional volume of the same buffer is added so that a total volume of 100 ml. is reached.

Example 9 1.5 g. of crushed, purified Amberlite IRA 411 the size of 1 to 3p. is dispersed in 5 ml. of tris-HCl-saline buffer solution (pH 8.2), followed by the addition of an optimal amount of fractionated anti-al-fetoglobulin and 0.01 g. patent blue. The system is shaken at 4 C. for 2 hours, whereby the anti-al-fetoglobulin is adsorbed. The system is centrifuged and the resulting anti-al-fetoglobulin-amberlite IRA411 is centrifuged twice, using the same buflFer solution as above to which 0.5 w./v.% of normal rabbit serum and 0.1 w./v.% sodium azide have been added. After washing, the sediment is dispersed in about 90 ml. of the same buffer solutionas above and brought to an exact pH value of 8.5. An additional volume of the same buffer solution is used to make up the dispersion to a total volume of 100 ml.

Example 10 0.25 g. of polystyrene latex the size of 0.8a is dispersed in 8 ml. of sodium chloride-phosphate buffer solution (pH' 8.5) followed by the addition of an optimal amount of anti-al-fetoglobulinserum. The system is shaken at 37 C. for 1 hour, whereby the anti-al-fetoglobulin 'is adsorbed onto'the carrier medium. The system is centrifuged and the resulting anti-al-fetoglobulin-latex is centrifuged twice, using the same buffer as above to which 0.1 w./v.% sodium azide has been added. After washing, the anti-a1- fetoglobulin-latex is dispersed in about 90 ml. of the same buffer solution as above and the dispersionbrought to pH 8.5. An additional, volume'of the same buffer is used to make up the dispersion to a total volume of 100ml.

Example 11 "Kaoline is purified by fractional precipitation to the size range of l to 3 1. and 2 grams of the purified kaolin is dispersed in 10 mi. of sodium chloride-borate buffer solution, followed by the addition of an optimal amount of solutionto which 0.5 w./v.% Tween so and 0.01; w.' thimeros'al have been added. To this system. is further.

added about 90 ml. ofthe' same buffer asaboveso that,

the pH of the system if 615. Finally, an additionalamount of the same buffer is used to make up the system to 'a total volume of 100 ml.

' globulin serum.

PREPARATION OF ANTI-eI-FETOGLOB UL IN Example 12 To 1 part (by weight) of the serum of a patient having a primary hepatoma is added by dissolution in ethanol. The 28' v./v.% ethanolic solution is adjusted to pH 6.4 and allowed to stand at 5 C. for 14 hours. The solution is centrifuged and the supernatant liquid is removed.,A 0.02 M solution of barium acetate is added to the superv natant liquid and the mixture is made into a 25 v.'/v.% ethanolic solution, which is adjusted to pH 6.7. The solution is allowed tostand at -5 C. for 2 hours, at the end of which time it is centrifuged. The supernatant is made into a 40 v./v.% ethanolic solution which is further allowed to stand at --5 C. for 2 hours. The solution'is centrifuged and the supernatant is made into a 40 v./v.%' ethanolic solution which is left standing at l( )v C. for 14 hours, at the end of which time-the resulting precipitate is recovered. The precipitate is dissolved in a solution of sodium citrate and the solution is dialyzed against water at 4 C. for a few days, at the end of which time the dialyzate is lyophilized to obtain the antigen.

A predetermined amount of this antigen is used to immunize rabbits by the complete adjuvant method and when the antigen titers have reached the desired level, the rabbits are bled and the sera separated. To those sera is added 0.2 volume of normal human serum and the mixture is allowed to stand at 37 C. for 1 hour and, then, at 4 C. overnight. The system is then centrifuged to obtain the desired anti-al-fetoglobulin serum.

The same procedure as mentioned above is conducted except using an immunoadsorbent instead of human serum to obtain the same result.

The immunoadsorbent is prepared as follows:

10 ml. of the normal human serum is adjusted to pH 5.5 with an acetate buffer solution. There is added-1.0 ml.- of glutaraldehyde and the solution is allowed to stand for 3 hours. After adding a phosphate buffer solution, the solution is centrifuged and the precipitants collected.

The precipitants are washed with the above buffer solution several times and suspended with the above buffer solution.

Example 13 To 1 part of the serum of a patient is added 1 part of 0.1 M sodium chloride-phosphate buffer solution (pH' 7.2), followed by the gradual addition of 2 parts of a saturated aqueous solution of ammonium sulfate while stirring, whereupon a precipitate is formed. The system is allowed to stand overnightat 4 C. and is then centri-' fuged at 8,000 r.p.m. for 20 minutes. The supernatant is dialyzed against water at 4 C..for afew days, at the end of which time the dialyzate is lyophilized to obtain the desired antigen. e v

This antigen was used to immunize a goat, followed by the same treatments asdescribed in Example 12. The resulting antiserum was absorbed with 0.35 volume ofobtain the desired anti-al-feto-.-

normal human serum "to Example 14- 5 milliliter sampleof the ascites ,of 1. patientihaviug, a primary, hepatoma is run into acolumnj, (3 100, cm.)

(second peak) to albumin (third peak) are arranged by Ouchterlonys method to confirm the al-fetoglobulin fraction, which is collected. This fraction is dialyzed using water as an external phase at 4 C. for a few days and the dialyzate is concentrated under suction using a collodione bag to obtain the desired antigen.

This antigen is used to immunize rabbits, followed by treatments similar to those described in Example 12. The resulting antiserum is absorbed with 0.2 volume of normal human serum to obtain the required anti-al-fetoglobulin serum.

Example 15 The antigen obtained according to Example 14 is further. purified by means of an isoelectric fractionation apparatus. Thus, the antigen is run onto a sucrose-gradient column containing 0.5 v./v.% carrier ampholyte of pH 3-10. While the column is maintained at 4 C., a. current of 700 volts is applied for 2 days. After this separation, the fractions of pH 4.2 to 4.5 are pooled and dialyzed at 4 C. for a few days, using water as the external fluid. The dialyzate is then concentrated under suction using a collodione bag to obtain the desired antigen.

This antigen was used to immunize rabbits, which were treated in the same manner as in Example 12. The resulting antiserum is absorbed with 0.1 volume of normal human serum to obtain the desired anti-al-fetoglobulin serum.

Example 16 A 50 ml. sample of the ascites of a patient having a primary hepatoma is diluted with water to 4 times its initial volume and the diluted sample is dialyzed against 5 liters of 0.1 w./v.% solution of acrynol for 24 hours. The acrynol solution is centrifuger at 4,000 r.p.m., and

the sediment is dissolved by shaking in 50 ml. of physio- 3 logical saline solution. The solution is centrifuged a second time at 4,000 r.p.m. The supernatant liquid is dialyzed against an acetate buffer solution (ionic strength 0.04, pH 5.0) and the dialyzate is run into a column of carboxymethyl-cellulose (pH 5.0, ionic strength 0.04), which is eluted to 0.2 M. Since al-fetoglobulin starts eluting at about 0.055 M, this fraction is collected. The above procedure is repeated twice to obtain a single al-fetoglobulin fraction.

Whereas the al-fetoglobulin titer of the original ascites is 3146 U/ml. (50 ml.), the final titer is 600 U/ml. (200 ml.) which represents a recovery of 70 w./v.%. Since the original amount of albumin was 7.623 mg./ml., (50 m1), it is impossible to detect the same with the immune-plate at the final stage.

Example 17 A 50 m1. sample of the ascites of a patient having a primary hepatoma is diluted with water to 4 times its original volume and dialyzed against 5 liters of 0.5 w./ v.% solution of acrinol for 24 hours. The dialyzate is centrifuged at 3,000 r.p.m. and the sediment is dissolved by shaking in 50 ml. of physiological saline solution. The solution is centrifuged a second time at 3,000 r.p.m. The supernatant liquid is dialyzed against tris-HCl-saline buffer (0.03 M, pH 4.5) and the dialyzate is run onto a column of DEAE-cellulose (pH 4.5, ionic strength 0.04) and eluted to 0.2 M. The eluate is similarly treated with a column of CM-cellulose to obtain a single al-fetoglobulin fraction.

Whereas the ul-fetoglobulin titer of the original ascites is 2,500 U/ml. (50 ml.), the final titer is 400 U/ml. (180 ml.), which represents a recovery of 60 w./v.%.

Incidentally, while the original amount of albumin is 7.915 mg./ml., it is impossible to detect it with the immuno-plate at the final stage.

Having now fully described the invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention.

Accordingly, what is claimed and intended to be covered by letters patent of the United States is:

1. An immunological gel, diagnostic agent for primary hepatoma, which comprises: water; from 1 to 10 w./ v.% anti-al-fetoglobulin serum, prepared by immunizing an animal with ascites from a patient having a primary hepatoma; from 1 to 15 w./v.% of a immunological gel support medium; a quantity of surfactant sufiicient to reduce the percentage of doubtful positive results attributable to hypercholesteric sera in clinic applications; a coloring agent; a preservative; and a buifering agent in an amount to buffer the gel to a pH range of 6.5 to 8.5.

2. The diagnostic agent of claim 1, wherein the immunological gel support medium is selected from the group consisting of agar, dextrin, starch and pectin.

3. The diagnostic agent of claim 1, wherein the immunological gel support medium is gelatin or a polyacrylamide gel.

4. The diagnostic agent of claim 1, wherein the surfactant is selected from the group consisting of polyoxyethylenesorbitan monooleate, sodium oleate, polyoxyethylenesorbitan monolaurate and saponin.

5. The diagnostic agent of claim 1, wherein the surfactant is taurocholic acid, which is present in an amount from 0.2 to 0.5 w./v.%.

6. The diagnostic agent of claim 1, wherein the coloring agent and the preservative are present in amounts of from 0.001 to 0.005 w./v.% and 0.1 w./v.%, respectively.

References Cited UNITED STATES PATENTS 3,088,875 5/1963 Fisk 424--12 3,551,555 12/1970 Schuurs 424-12 3,553,310 1/1971 Csizmas 424-12 X 3,641,235 2/1972 Weiss 424-12 X OTHER REFERENCES Alpert, The New Eng. J. Med., vol. 278, May 2, 1968, pp. 984-986.

Endo, Igaku no ayumi, vol. 68, N0. 11, 1969, pp. 519- 531.

Oshiro, Dissertation, microfilm, 67-13,037, Univ. Microfilms, Ann Arbor, Mich., 1967, pp. 31-33, 73, 142, 143.

Gitlin, J. Clin. Invest, vol. 45, No. 11, 1966, pp. 1826- 1837.

Bozicevich, PSEBM, vol. 97, 1958, pp. 180-183.

Difco, Supplementary Literature, Difco Labs, Detroit, November 1968, p. 166.

Kwapinski, Methods in Serol. Res., John Wiley & Sons, N.Y., 1965, pp. 170-172.

Mancini, C-hem. Abs., vol. 63, 1965, p. 18846.

ALBERT T. MEYERS, Primary Examiner A. P. FAGELSON, Assistant Examiner U.S. Cl. X.R. 42412, 

